The most primitive hematopoietic stem cells (HSC) will reconstitute all of the hematopoietic lineages for an entire lifespan. These pluripotent hematopoietic stem cells (PHSC) are the transplantable cells that are ultimately the targets for gene delivery in stem cell-based gene therapies. One defining characteristic for PHSC is that they will survive most cytoablative conditioning regimens. The mechanisms for their resistance to these toxic agents suggest potential strategies by which these cells can be selected in vitro. One mechanism for drug resistance lies in the ability to efflux toxic substances out of the cell via the multiple drug resistance (MDR) pump. Fluorescent substrates for the MDR pump have permitted the isolation of PHSC based on their high capacity for dye efflux in a variety of assay systems. Drug resistances may also be conferred by more specific mechanisms. For example, a cytosolic aldehyde dehydrogenase (ALDH) mediates resistance to cyclophosphamide (CPA), an alkylating agent used in cytoreductive regimens in preparation for bone marrow transplant. Thus, expression of ALDH can be considered a selectable marker for true PHSC.
The therapeutic effectiveness of CPA has been attributed largely to the ability of PHSC and intestinal crypt cells to survive the drug regimen. Human hematopoietic progenitors express a cytosolic ALDH and primitive human HSC derived from mobilized peripheral blood stem cells can be selected when placed in culture with cyclophosphamide for 7 days. Jones et al. have demonstrated that long-term reconstituting murine PHSC can be isolated by providing a membrane-permeable fluorescent substrate for ALDH and by then selecting cells with the highest levels of ALDH activity (Jones (1995) Blood 85:2742; Jones et al. (1996) Blood 88:487). In these studies, dansyl aminoacetaldyde (DAAA) was used to stain murine bone marrow cells prepared by countercurrent elutriation.
Preliminary studies using DAAA indicate that this reagent is unusable on preparations of human hematopoietic cells because the signal intensity of the reagent is too high to resolve discrete cell populations by flow cytometry. The present invention provides a fluorescent ALDH substrate that is free of the problems associated with DAAA and that can be used in the purification of primitive human hematopoietic cells.